Asymmetric Bidirectional Transcription from the FSHD-Causing D4Z4 Array Modulates DUX4 Production

Facioscapulohumeral Disease (FSHD) is a dominantly inherited progressive myopathy associated with aberrant production of the transcription factor, Double Homeobox Protein 4 (DUX4). The expression of DUX4 depends on an open chromatin conformation of the D4Z4 macrosatellite array and a specific haplotype on chromosome 4. Even when these requirements are met, DUX4 transcripts and protein are only detectable in a subset of cells indicating that additional constraints govern DUX4 production. Since the direction of transcription, along with the production of non-coding antisense transcripts is an important regulatory feature of other macrosatellite repeats, we developed constructs that contain the non-coding region of a single D4Z4 unit flanked by genes that report transcriptional activity in the sense and antisense directions. We found that D4Z4 contains two promoters that initiate sense and antisense transcription within the array, and that antisense transcription predominates. Transcriptional start sites for the antisense transcripts, as well as D4Z4 regions that regulate the balance of sense and antisense transcripts were identified. We show that the choice of transcriptional direction is reversible but not mutually exclusive, since sense and antisense reporter activity was often present in the same cell and simultaneously upregulated during myotube formation. Similarly, levels of endogenous sense and antisense D4Z4 transcripts were upregulated in FSHD myotubes. These studies offer insight into the autonomous distribution of muscle weakness that is characteristic of FSHD

Loss of Maternal CTCF Is Associated with Peri-Implantation Lethality of Ctcf Null Embryos

CTCF is a highly conserved, multifunctional zinc finger protein involved in critical aspects of gene regulation including transcription regulation, chromatin insulation, genomic imprinting, X-chromosome inactivation, and higher order chromatin organization. Such multifunctional properties of CTCF suggest an essential role in development. Indeed, a previous report on maternal depletion of CTCF suggested that CTCF is essential for pre-implantation development. To distinguish between the effects of maternal and zygotic expression of CTCF, we studied pre-implantation development in mice harboring a complete loss of function Ctcf knockout allele. Although we demonstrated that homozygous deletion of Ctcf is early embryonically lethal, in contrast to previous observations, we showed that the Ctcf nullizygous embryos developed up to the blastocyst stage (E3.5) followed by peri-implantation lethality (E4.5–E5.5). Moreover, one-cell stage Ctcf nullizygous embryos cultured ex vivo developed to the 16–32 cell stage with no obvious abnormalities. Using a single embryo assay that allowed both genotype and mRNA expression analyses of the same embryo, we demonstrated that pre-implantation development of the Ctcf nullizygous embryos was associated with the retention of the maternal wild type Ctcf mRNA. Loss of this stable maternal transcript was temporally associated with loss of CTCF protein expression, apoptosis of the developing embryo, and failure to further develop an inner cell mass and trophoectoderm ex vivo. This indicates that CTCF expression is critical to early embryogenesis and loss of its expression rapidly leads to apoptosis at a very early developmental stage. This is the first study documenting the presence of the stable maternal Ctcf transcript in the blastocyst stage embryos. Furthermore, in the presence of maternal CTCF, zygotic CTCF expression does not seem to be required for pre-implantation development

CTCF Haploinsufficiency Destabilizes DNA Methylation and Predisposes to Cancer

Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf+/− tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf+/− tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression

<i>Ctcf</i> nullizygous mice display early embryonic lethality.

*<p>Control crosses.</p><p>NA = not applicable.</p

<i>Ctcf</i> nullizygous embryos can develop <i>ex vivo</i> to the morula/blastocyst stage.

<p><i>Ctcf</i> nullizygous embryos can develop <i>ex vivo</i> to the morula/blastocyst stage.</p

Analysis of E5.5 and E6.5 <i>Ctcf</i> embryos.

<p>NA = not applicable.</p

<i>Ctcf</i> maps to mouse chromosome 8.

<p>The Jackson Laboratory interspecific backcross panels (BSS and BSB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034915#pone.0034915-Rowe1" target="_blank">[28]</a> were utilized to map the <i>Ctcf</i> locus to mouse chromosome 8. The loci are listed in order with the most proximal at the top. The black boxes represent the C57BL6/JEi allele and the white boxes the SPRET/Ei allele. The number of animals with each haplotype is given at the bottom of each column of boxes. The percent recombination (R) between adjacent loci is given to the right of the lower figure together with the standard error (SE) for each R.</p

<i>Ctcf</i> nullizygous embryos fail to develop beyond the E3.5 stage and undergo apoptosis.

<p>(<b>A</b>) <i>Ex vivo</i> outgrowth of E3.5 embryos. Blastocysts (E3.5) were isolated from <i>Ctcf</i> heterozygous (+/−) intercrosses and cultured <i>ex vivo</i> for the indicated number of days. At the end of the culture period the embryos were harvested and PCR genotyping and RT-PCR performed. The cultured <i>Ctcf</i> (+/+) and (+/−) embryos outgrew to form an inner cell mass (ICM) and trophoectoderm (TE) while little proliferation was noted in the cultured <i>Ctcf</i> (−/−) embryos (40×). (<b>B</b>) E3.5 blastocyst stage embryos isolated from the indicated crosses were subjected to DAPI staining and anti-CTCF immunochemistry (40×). All (16/16) embryos analyzed from the <i>Ctcf</i> (+/−) heterozygous intercrosses were positive for CTCF protein expression, displaying staining patterns similar to the two representative embryos shown here. The expected Mendelian ratio of genotypes in such crosses indicates that the chance that none of these 16 embryos were <i>Ctcf</i> (−/−) is less than 0.05 (i.e. (3/4)¹⁶≅0.01). (<b>C</b>) Day two outgrowths of E3.5 embryos of the indicated genotype. Blastocysts (E3.5) were cultured <i>ex vivo</i> for two days and then subjected to DAPI staining, anti-CTCF immunohistochemistry and TUNEL assay (40×). <i>Ctcf</i> (+/+) and (+/−) embryos outgrew and expressed CTCF, while cultured <i>Ctcf</i> (−/−) embryos failed to outgrow and exhibited loss of CTCF protein expression and increased apoptosis. The observations displayed here involve one <i>Ctcf</i> (+/+) and two <i>Ctcf</i> (−/−) embryos cultured <i>ex vivo</i> and are representative of over 60 embryos that were analyzed from <i>Ctcf</i> (+/−) heterozygous intercrosses.</p

Generation of <i>Ctcf</i> knockout mice.

<p>(<b>A</b>) A schematic diagram of the mouse <i>Ctcf</i> locus and the targeting vector derived from the wild type <i>Ctcf</i> allele as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034915#s4" target="_blank">Materials and Methods</a> are shown. E₁–E₁₂ denote <i>Ctcf</i> exons 1 through 12. Restriction enzyme sites shown on map are as follows: E denotes <i>Eco</i>RI, X denotes <i>Xba</i>I, and S denotes <i>Spe</i>I. The locations of both the 5-prime and 3-prime <i>Ctcf</i> genomic probes for the Southern blot analysis are indicated. Red arrows show the locations of genotyping primers. A schematic diagram of genomic digest with <i>Eco</i>RV of the <i>Ctcf</i> knock-out and wild type alleles is shown below. (<b>B</b>) A Southern blot analysis of <i>Eco</i>RV digested genomic DNA utilizing the 5-prime and 3-prime <i>Ctcf</i> genomic probes distinguishes the wild type (14 kb or 25 kb) from the mutated (8 kb or 13 kb) <i>Ctcf</i> alleles. (<b>C</b>) FISH analysis of chromosome spreads from lymphocyte cultures derived from <i>Ctcf</i> heterozygous (+/−) and wild type (+/+) mice. The probe for <i>Ctcf</i>, a lambda phage clone 19.1 containing a 17 kb <i>Ctcf</i> genomic DNA insert that includes all coding <i>Ctcf</i> exons that were deleted in the <i>Ctcf</i> knockout allele (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034915#pone-0034915-g002" target="_blank">Figure 2A</a>), was visualized with fluorescein (green fluorescence). The probe for chromosome 8 identification, a BAC clone (MB11301, Research Genetics), was visualized with Cy3 (red fluorescence). Examples of single metaphase cells from the <i>Ctcf</i> (+/−) and <i>Ctcf</i> (+/+) mice after hybridization to the <i>Ctcf</i> and chromosome 8 probes are shown. Both homologues of chromosome 8 are labeled with a red and a green signal in the wild type mouse, whereas absence of green signals on one homologue of chromosome 8 confirmed the presence of a deletion of <i>Ctcf</i> in the <i>Ctcf</i> (+/−) mouse.</p